Mate choice, copulation, genital morphology, and sperm storage are not very well understood in millipedes. The use of three-dimensional x-ray computed tomography (µCT) provides new morphological data regarding millipede reproductive systems in both the female and male, including chitinous sclerites and membranes, muscles, glands, oviducts, and sperm conduits. Here we present a complete integrated account of the morphology and function of the female genital organs in the family Polydesmidae (Diplopoda: Polydesmida) using µCT, UV fluorescence imaging, and scanning electron microscopy. These data allow us to consider competing hypotheses regarding millipede vulva formation. We additionally present the morphology of copulatory interface in Pseudopolydesmus Attems, 1898 using images of a mating pair in copula and by simulating the interface of the organs using 3D models from µCT, allowing us to tentatively identify a lock-and-key-like mechanism. Finally, we use µCT to reveal the topology of the seminal canal in the gonopod of male Pseudopolydesmus, a topic that has remained unresolved for nearly 80 years.

Specimens were dehydrated successively in a series of 80%, 85%, 90%, and finally 95% ethanol (unless already stored in 95% ethanol) with at least 2 hours in each stage. Specimens were stained for 24-48 hours in a 1% solution of iodine in 100% ethanol (after Metscher, 2009). The iodine stains soft tissues (Gignac et al., 2016), increasing their contrast under x-ray imaging.

Dehydrated and stained specimens were mounted in 95% ethanol in either a 0.5 mL or 1.0 mL microfuge tube depending on their size. Intact full body specimens were mounted head-first and gently wedged in place (to prevent movement during imaging) using a small layer of cotton. Body ring fragments were mounted between two cotton layers.

3D image acquisition was performed using an XRadia microXCT-200 housed in the Center for Molecular and Genomic Imaging at the University of California, Davis. Images were acquired using detectors with 4x or 10x magnification (producing images with 4.5 µm and 2.0 µm voxel resolution, respectively). Specifications for each scan are provided in the original publication. The 3D images were reconstructed as .txm files, which were converted to .tif stacks using Drishti Import (Limaye, 2012).

3D image files (.txm) can be viewed using Amira/Aviso. Alternatively, the free program Drishti Import (Limaye, 2012) can be used to preview the stack and convert it to the Metaimage format (.mhd). Metaimage files can be viewed in the free program ITK-SNAP (Yushkevich et al., 2006).